The present invention relates to microscopic digital imaging of complete tissue sections for medical and research use. In particular it describes a method for maintaining high quality focus during high throughput montage imaging of microscope slides.
Laboratories in many biomedical specialties, such as anatomic pathology, hematology, and microbiology, examine tissue under a microscope for the presence and the nature of disease. In recent years, these laboratories have shown a growing interest in microscopic digital imaging as an adjunct to direct visual examination. Digital imaging has a number of advantages including the ability to document disease, share findings, collaborate (as in telemedicine), and analyze morphologic findings by computer. Though numerous studies have shown that digital image quality is acceptable for most clinical and research use, some aspects of microscopic digital imaging are limited in application. Perhaps the most important limitation to microscopic digital imaging is a xe2x80x9csub-samplingxe2x80x9d problem encountered in all single frame images. The sub-sampling problem has two components: a field of view problem and a resolution-based problem. The field of view problem occurs when an investigator looking at a single frame cannot determine what lies outside the view of an image on a slide. The resolution-based problem occurs when the investigator looking at an image is limited to the resolution of the image. The investigator cannot xe2x80x9czoom inxe2x80x9d for a closer examination or xe2x80x9czoom outxe2x80x9d for a bird""s eye view. Significantly, the field of view and resolution-based problems are inversely related. Thus, as one increases magnification to improve resolution, one decreases the field of view.
To get around the limitation of single frame imaging, several neighboring images can be tiled together to form a montage image or xe2x80x9cvirtual slidexe2x80x9d. To accomplish this, a robotic microscope systematically scans the entire slide, taking an image at every field. The individual images are then xe2x80x9cknittedxe2x80x9d together in a software application to form a very large data set with very appealing properties. The robotic microscope can span the entire slide area at a resolution limited only by the power of the optical system and camera. Software exists to display this data set at any resolution on a computer screen, allowing the user to zoom in, zoom out, and pan around the data set as if using a physical microscope. The data set can be stored for documentation, shared over the Internet, or analyzed by computer programs.
The xe2x80x9cvirtual slidexe2x80x9d option has some limitations, however. One of the limitations is file size. For an average tissue section, the data generated at 0.33 um/pixel can be between two and five gigabytes uncompressed. In an extreme case, the data generated from one slide can be up to thirty-six gigabytes.
A much more difficult limitation with the prior systems is an image capture time problem. Given an optical primary magnification of twenty and a two-third inch CCD, the system field of view is approximately (8.8 mmxc3x976.6 mm)/20=0.44xc3x970.33 mm. A standard tissue section of approximately 2.25 square centimeters, therefore, requires approximately fifteen hundred fields to cover the tissue alone.
Field rate in montage systems is limited by three factorsxe2x80x94camera frame rate, image processing speed, and the rate of slide motion between fields. Given today""s technology, the rate of slide motion is a significant limiting factor largely because the existing imaging systems require the slide to come to a stop at the center of each field to capture a blur free image of the field.
The three dimensional characteristic of the tissue sample and the slide places additional limitations on the imaging system. Like all lenses, microscope optics have a finite depth of field, the distance within which objects will appear to be focused. A typical depth of field is about 8 microns for a 10xc3x97objective, and in general, as the magnification increases, the depth of field decreases. While microscope slides are polished glass, the flatness of the slide can vary on the order of 50 microns or more across the slide. The variations in the tissue sample thickness and any defects associated with placing the sample on the slide such as folds in the tissue, also affect the optimal position of the slide with respect to the imaging optics. The magnitude of the optimal position and the limited depth of field of the microscope optics require the focus to be adjusted as the system moves from field to field. In order to determine the optimal focal position, multiple images must be acquired as the slide is displaced along the optical axis (perpendicular to the scanning plane) and a quantitative value such as contrast calculated for each image. The direction and the amount of displacement required to maintain high-quality focus is dependent on the three dimensional structure of the slide and tissue specimen. Given that the average tissue section mentioned above requires fifteen hundred image fields, the time required to refocus at each tile can contribute substantially to the overall scan time.
Thus, a system is needed to reduce the overhead associated with refocusing while maintaining efficiency and image quality. This invention relates to maintaining high-quality focus as it is scanned without having to rely on refocusing during the scanning process.
Accordingly the invention relates to a method and system for ensuring that a scanning process captures a high-quality montage image of a slide by enabling accurate focus control of raw image tiles of the slide. The system includes a focus point selection component, a focal surface determination component, and a scan control component. The focus point selection component evaluates tissue regions of a thumbnail image and selects several points to initially focus microscope optics on a point-by-point basis. The focal surface determination component uses focus positions to generate a three-dimensional data set corresponding to optical specimen distance at each stage location, wherein data points in the data set are used as input to a routine that generates control parameters for a slide scanning process. The scan control component captures the high-quality montage image by maintaining motion of a stage and synchronization of a microscopic imaging system during montage image acquisition. The scan control component thus, enables accurate focus control of optical elements without requiring stopping and refocusing of the stage at each tile location and substantially reduces montage acquisition time. The system also includes means for placing focus positions from the focus point selection component into microscope optics in the focal surface determination component and for passing tissue information and surface parameters to the scan control component.
It is therefore an object of the invention to provide an automated, microscopic imaging system for whole slide montage in which standard microscope optics, an off-the-shelf camera and a simple motorized stage can be used to produce perfectly aligned, well-focused image tiles, and acquire these images at a speed limited by the camera frame rate. The present invention uses the fact that the scale length associated with the three dimensional nature of the slide and tissue specimen is large compared to the field of view of a single camera image. By pre-sampling key regions on the slide, a focal surface can be calculated, control parameters generated based on this surface, and the optimal stage position with respect to the optical elements maintained throughout the scanning process, insuring high-quality images with minimal overhead.
Additional features and advantages of the invention will be set forth in the description that follows, and in part will be apparent from the description, or may be learned by practice of the invention. The objectives and advantages of the invention to be realized and attained by the microscopic image capture system will be pointed out in the written description and claims hereof as well as the appended drawings.